1. Field of the Invention
The present invention relates to novel recombinant Sendai virus vector constructs for the reprogramming of differentiated somatic cells into induced pluripotent stem (iPS) cells
2. Description of the Related Art
Along with the progression toward an aging society, diseases caused by tissue degeneration and damage are increasing rapidly. For example, diseases that increase in frequency with age include metabolic syndromes, such as cerebral infarction, cardiac infarction and renal failure, as well as diseases caused by age-related tissue degeneration, such as Alzheimer's disease, Parkinson's disease and osteoporosis. In addition, type I diabetes, multiple sclerosis, chronic rheumatoid arthritis, thermal burn, spinal damage from injury, and genetic diseases caused by congenital abnormalities in the genetic code, are all diseases caused by tissue degeneration and damage. A number of regeneration therapies are being developed as a means for treating these diseases.
Regeneration therapies can be tentatively classified into two groups: (1) guided regeneration therapies that target the activation of tissue stem cells residing in a patient's tissue, and (2) cell replacement therapies requiring the transplantation of exo-vivo generated stem cells or stem-cell-derived somatic cells or tissues, into a patient. The regeneration potential of tissue stem cells is however often limited. Development of more effective cell replacement therapies is therefore essential to the practical application of regeneration therapies. In particular, with regard to genetic diseases, cell replacement therapies are contemplated where a patient's cells are genetically engineered ex-vivo to repair or replace defective genes prior to transplantation back into the patient.
Treatment of diseases caused by tissue degeneration/damage, also requires the preparation of large amounts of stem cells or stem cell-induced somatic tissues. Thus, pluripotent stem cells capable of self-renewal over long periods of time while maintaining their differentiation potential into various tissue types are essential requirements for the development of effective cell replacement therapies. To date only a few pluripotent stem cells have been characterized that meet these requirements and include embryo-stem cells (ES cells) derived from the epiblast of early mammalian embryos, and ES cells derived from primordial germ cells. These heterogenic cells cannot be used in cell replacement therapies however because their genetic information is different from that of a patient's hence transplantation of the cells into a patient would inevitably lead to tissue rejection.
Cell replacement therapies therefore require the generation of isogenic pluripotent stem cells that are genetically identical to a somatic cell of a patient in order to avoid immunological rejection after transplantation into a patient. To obtain such cells, the invention contemplates the isolation and modification of a patient's own tissue cells to generate isogenic pluripotent stem cells to be practical and efficient, the procedure ideally will require only minimal surgical intervention to harvest a small sample of tissue cells. For example, the procedure contemplates the collection of easily accessible cells requiring minimal surgical intervention, e.g., skin fibroblast cells, oral mucosal cells or hair follicle epithelial cells. To avoid undue stress and discomfort to the patient, the procedure further contemplates the collection of no more than approximately 104 cells for the generation of isogenic pluripotent stem cells.
It is known from research on human ES cells that extensive culture of pluripotent stem cells over long periods of time inevitably leads to the appearance within the cell population of chromosomal abnormalities, such as chromosomal deletions, amplifications and translocations. If established pluripotent stem cells are heterogeneous with respect to chromosomal stability, they would require continuous selection for cell lines with minimal chromosomal rearrangements which would be time consuming, expensive and inefficient. Thus, the method of generating pluripotent stem cells of the invention should reproducibly select not only for pluripotency but also for chromosomal stability within a selected homogeneous clonal population. One method of determining uniformity within a selected clonal population would be to determine an index comprising a correlation coefficient between respective gene expression patterns of the selected cell lines. Using such a selection criteria, only clones of pluripotent cells approaching a coefficient of 1, preferably equal to or greater than 0.98 would be deemed to be sufficiently stable for use in cell replacement therapies.
After the isolation of stable clonal populations derived from a patient's somatic cells, the pluripotency potential and ability to differentiate into various tissues is then determined.
Pluripotency can be verified by assessing the potential for differentiation in vitro or by determining the degree of differentiation in vivo after transplantation of the candidate pluripotent stem cells into an immunodeficient animal. Another caveat to this approach is the propensity of pluripotent cells to form malignant teratocarcinomas after transplantation. Thus, in view of the need to verify that a candidate pluripotent stem cell line is not only pluripotent but also safe and suitable for use in regeneration therapies, verification based on differentiation within malignant teratocarcinomas is inappropriate (see Nakanishi, Regenerative Medicine, 9, 216-221, 2010)
One way to distinguish between a pluripotent stem cell which is less likely to become a malignant tumor in vivo and thereby safe for human therapy, from a teratocarcinoma which is a malignant tumor with differentiation capability, is to analyze germ-line (germinal) transmission in a laboratory animal such as a mouse i.e., to analyze the transmission of genetic information derived from the pluripotent stem cell to the germ line within a chimeric animal created from pluripotent cells. Using this method, germ-line transmission is only observed with pluripotent stem cells that are less likely to become a malignant tumor after transplantation in vivo and are thereby safe for human therapies. Germ line transmission is not observed with pluripotent cells that form a teratocarcinoma after transplantation. However, this verification obviously cannot be performed in a human.
To establish reproducible protocols for the isolation and selection of pluripotent stem cells which are safe for human therapy trials, it is first necessary to identify appropriate pluripotent stem cells from a laboratory animal using the germ line transmission method. In other words, protocols for establishing therapeutically safe pluripotent stem cells first need to be developed using non-human animals, preferably, the mouse for which reproductive technologies are well established. The procedures required for efficient germ line transmission of pluripotent stem cells in mice can then be extrapolated to human therapeutic applications.
From the above discussion, a method of reproducibly generating human pluripotent stem cells applicable to regeneration therapies needs to meet the following requirements: 1) an established human pluripotent stem cell must be genetically identical to that of a patient's cell; 2) a human pluripotent stem cell must be established from 104 somatic cells or less; 3) established pluripotent stem cells must be clonal and genetically stable; and 4) the germ-line transmission must be verifiable using a chimeric animal derived from pluripotent stem cells and establishing that the pluripotent stem cells contribute to the germ line.
A pluripotent stem cell having genetic information identical to that of a patient, can be generated by introducing a specific combination of pluripotentency-inducing genes into human somatic cells using retroviral vectors. Ectopic expression of these genes results in the generation of a human induced pluripotent stem cell (human iPS cell) closely resembling a human ES cell. For example, the introduction and the expression of Oct3/4, Sox2, Klf4 and c-Myc in human skin fibroblast cells using a retroviral or lentiviral vector results in the transformation of a somatic cell into a human iPS cell (see Takahashi, et al., Cell, 131, 861-872, 2007). Similarly, the introduction and expression of Oct3/4, Sox2, Nanog and LIN28 in human skin-derived fibroblast cells using lentiviral vectors results in the generation of human iPS cells (see Yu, et al., Science, 318, 1917-1920, 2007).
Furthermore, a human iPS cell can also be produced using a modified technique in which one or two of the above four types of genes are substituted by a low-molecular-weight compound. For example, one publication reports that the introduction and expression of two genes, Oct3/4 and Sox2, into normal human skin-derived fibroblast cells cultured in the presence of a histone deacetylase inhibitor results in the transformation of the fibroblast cells into human iPS cells (see Huangfu, et al., Nature Biotechnology, 26, 1269-1275, 2008).
However, in each of the above methods, the genes introduced into a somatic cell are known to be inserted randomly in the iPS cell's host DNA. Genetic information within the iPS cell is therefore different from that of the original skin fibroblast cell. This means that an iPS cell produced by the above techniques fails to meet the requirement that “therapeutically safe pluripotent stem cells have genetic information that is identical to that of a patient's cell.”
In terms of ensuring the safety of cell replacement therapy, the above gene insertion protocol causes the following problem. If exogenous genes are inserted into the chromosomes at random, they are likely to abnormally activate genes adjacent to the insertion sites and possibly cause unpredictable side effects, even promote the expression of genes involved in the initiation of tumor. For example, it is known that, if genes are inserted at non-specific positions on chromosomes of a human bone marrow stem cell that is capable of maintaining a self-renewal ability over a long period of time, the expression of tumorigenic genes, that is normally inhibited in these cells, can become transcriptionally active due to the proximal insertion of foreign genes, which can ultimately lead to a high frequency of tumor initiation in these cells (see Hacein-Bey-Abina, et al., Science, 302, 415-419, 2003).
This gene insertion phenomenon further causes the following problem in terms of ensuring the safety of the cell replacement therapy. In an iPS cell produced by inserting foreign genes into chromosomes, although expression of the foreign genes may be inhibited during the period where a cell is kept in an undifferentiated state, the expression of the foreign genes may be induced when the cell has differentiated into a somatic cell, and the resulting cell is likely to become cancerous. For example, it is known that, in an iPS cell-derived transgenic mouse produced by the introduction of Oct3/4, Sox2, Klf4 and c-Myc into a skin-derived normal fibroblast cell using retroviral vectors, cancer develops at high frequencies due to reactivation of the externally introduced c-Myc gene (see Okita, et al., Nature, 448, 313-317, 2007). Further, it is noted that expression of the Klf4 or Oct3/4 gene also has the potential to lead to the initiation of cancer (see Jaenisch and Young, Cell, 132, 562-582, 2008).
With a view to solving the above problems caused by random gene integration into the chromosomal DNA of the host somatic cell, researchers have attempted to introduce plasmid DNA into a somatic cell that is capable of only transient expression of the iPS-inducing genes. For example, one report describes lipofection of Oct3/4, Sox2, Klf4 and c-Myc, into a mouse skin-derived fibroblast cell that results in the transient expression of these genes sufficient to generate an iPS cell but without the concomitant insertion of the foreign genes into chromosomes of the host fibroblast cell (see Okita, et al., Science, 322, 949-953, 2008). However, close analysis of this method shows that the introduced genes were found to be inserted into chromosomes in 75% of the mouse iPS cells generated. Thus, this method does not necessarily prevent insertion of foreign genes into the genome of the iPS cell. Moreover, there is no report thus far indicating that a human iPS cell can be produced using this approach without insertion of foreign iPS-inducing genes into the genome of the host somatic cell.
In other efforts to mitigate the problems caused by gene insertion into the host cell's genome, transient expression of iPS-inducing genes (Oct3/4, Sox2, Klf4 and c-Myc) using adenoviral vectors was shown to transform a somatic cell into an iPS. For example, it has been reported that a mouse iPS cell can be produced by cloning Oct3/4, Sox2, Klf4 and c-Myc into adenoviral vectors and co-transducing the recombinant adenoviral vectors into a mouse liver-derived normal liver cell (see Stadfeld, et al., Science, 322, 945-949, 2008). However, here again, the introduction of adenoviral vectors into the host cell inevitably leads to the random insertion of the vectors into the genome of the host cell at significant frequencies (see Ohbayashi, et al., Proc. Natl. Acad. Sci. USA, 102, 13628-13633, 2005). Thus, this method fails to prevent random insertion of foreign genes into the genome of the iPS cell. Once again, there is no report indicating that a human iPS cell can be produced from a human-derived somatic cell, without insertion of foreign genes into the genome of the host cell.
It has also been reported that, after producing an iPS cell by randomly inserting Oct3/4, Sox2, Klf4 and c-Myc, into chromosomes, the inserted genes can then be excised from the host genome using recombinase (see Kaji, et al., Nature, 458, 771-775, 2009). For example, Cre recombinase has been shown to remove Oct3/4, Sox2, Klf4 and c-Myc genes from the host genome following induction of iPS cells (see Kaji, et al., Nature, 458, 771-775, 2009). However, at least in these reports, promoter regions necessary for inducing expression of the reprogramming genes remained in the host cell's genome. Genetic information of the produced iPS cell is therefore not identical to that of a parent cell, and interference with gene expression in proximity to the insertion site remains possible.
In other reports, the iPS inducing genes were introduced into the host somatic cells using a transposon. After transient expression of the iPS inducing genes, the transposon was removed by expression of transposase that excises the transposon and the covalently linked iPS inducing genes from the host cell's genome. (see Woltjen, et al., Nature, 458, 766-770, 2009). This method is, however, inefficient, because the probability of successful removal is only about 0.001% of the total number of produced iPS cells, and no examples using a human cell were given. According to a report by Woltjen, et al., even after removal of the transposable element by transposase, a residual four bases remain at the integration site. In this case, it cannot be denied that the potential for insertional mutagenesis remains possible. Moreover, the transposase used for removing the transposon is an enzyme having both an excision activity resulting in the removal of the transposon and an integrase activity that directs the insertion of transposons into the genome of the host cell. Thus, at least in theory, transposons excised from a particular insertion site may be re-inserted at a different location in the genome. Therefore, it would be necessary to check each iPS cell clone to verify that re-insertion has not reoccurred at a different location.
Yu, et al., have reported that simultaneous expression of Oct3/4, Sox2, Klf4, c-Myc, Nanog, LIN28 and SV40 T antigen in a human normal fibroblast cell, using an extrachromosomally-replicable circular DNA vector (EBV vector) having a replication origin of Epstem-Barr virus (EBV) and EBNA1 gene, transforms the fibroblast cell into an iPS cell. All foreign DNA in the iPS cell can then be eliminated by removal of the episome (see Yu, et al., Science, 324, 797-801, 2009). As of now, this is the only report describing the generation human iPS cells that are genetically identical to that of the parent somatic cell. However, the iPS cell production efficiency is only in the range of about 0.0003 to 0.0006%. Hence, at least 3×105 cells would be required to establish a single iPS cell. Moreover, EBV DNA is not only episomal but can also be inserted into the host cell's DNA at high frequency (see Hurley, et al., J. Virol, 65, 1245-1254, 1991). Thus, this method is also flawed because it does not prevent integration of foreign genes into the genome of the iPS cell. To verify the absence of integration of any foreign DNA again would require the screening of each iPS clone.
A number of different experimental strategies have been devised to generate a iPS cell having genetic information that is identical to that of the parent somatic cell. In one such attempt, a tissue cell nucleus was introduced into an enucleated oocyte (see Wakayama, et al., Science, 292, 740-743, 2001). In another attempt, a peptide capable of crossing cell membranes was fused to the N terminus of each of Oct3/4, Sox2, Klf4 and c-Myc, and scraped-loaded into somatic host cells (see Zhou, et al., Cell Stem Cell, 4, 381-384, 2009). However, there is currently no report indicating that a human iPS cell could be produced using any of these methods.
Recently, Fusaki, et al., (see Fusaki, et al., Proc. Jpn. Acad. Ser. B85, 348-362, 2009) reported a method designed to direct the expression of Oct3/4, Sox2, Klf4 and c-Myc genes in a human skin-derived fibroblast cell and generate pluripotent stem cells, using as a vector a Sendai virus that does not integrate into the host cell's genome (see Fusaki, et al., Proc. Jpn. Acad. Ser. B85, 348-362, 2009 and PCT/JP 2009/062911). In this report, iPS cells were established at a maximum efficiency rate of 1% by loading four types of reprogramming genes on individual vectors, mixing the vectors together and infecting a cell with the mixed vectors. However, this report makes no mention of the genetic stability and the clonality of the iPS cells. According to a semi-qualitative RT-PCR (Reverse Transcription-Polymerase Chain Reaction) analysis of the gene expression, it is immediately apparent that the established iPS cell lines were not entirely clonal i.e. they did not have identical characteristics with respect to chromosomal stability and gene expression profiles. Moreover, only a human iPS cell line is shown in the examples. The report therefore fails to demonstrate that this technique is broadly applicable to different animal species, and that germ-line transmission can be verified using a chimeric animal model derived from a iPS cell. To date, no method has been described for the generation of human iPS cells that are therapeutically safe for human regeneration therapies. There is therefore an unmet need for therapeutically safe iPS cells that fulfill the following four requirements: 1) the established human pluripotent stem cells have identical genetic information to that of the patient; 2) the human iPS cells can be generated from just 104 cells or less; 3) the established human iPS cells are clonal and genetically stable; and 4) the germ-line transmission can be demonstrated using a chimeric animal derived from a iPS cell.
Further, it has been reported that by using the above-described foreign genes, human iPS cells can be prepared from various tissue cells including skin-derived fibroblast cells (Takahashi, et al., Cell, 131, 861-872, 2007; Yu, et al., Science, 318, 1917-1920, 2007), hair root-derived keratinocytes (Aasen, et al., Nature Biotechnology, 26, 1276-1284, 2008), bone marrow-derived mesenchymal stem cells (Park, et al., Cell, 134, 877-886, 2008), neural stem cells (Kim, et al., Nature, 461, 649-653, 2009), adipose tissue-derived mesenchymal stem cells (Sun, et al., Proc. Natl. Acad. Sci. USA, 106 15720-15725, 2009), mesenteric cells (Li, et al., Cell Reprogram., 12 237-247, 2010), dental pulp cells (Oda, et al., J. Biol. Chem., 285, 29270-29278, 2010), tooth root cells (Egusa, et al., PLos One, 5, e12743, 2010), peripheral blood-derived mononuclear cells (Loh, et al, Cell Stem Cell, 7, 15-19, 2010; Staerk, et al., Cell Stem Cell, 7, 20-24, 2010), peripheral blood-derived T cells (Loh, et al, Cell Stem Cell, 7, 15-19, 2010; Staerk, et al., Cell Stem Cell, 7, 20-24, 2010; Seki, et al., Cell Stem Cell, 7, 11-14, 2010), and hematopoietic progenitor cells (Loh, et al., Blood, 113 5476-5479, 2009).
It is desirable that living human tissue cells as a raw material for human iPS cells are capable of being collected by a method which does not give a strong invasion into the human body and is free from the risk of contamination with microorganisms (bacteria and viruses). Among the above-listed human tissue cells, skin-derived fibroblast cells, bone marrow-derived mesenchymal stem cells, neural stem cells, adipose tissue-derived mesenchymal stem cells, mesenteric cells, dental pulp cells and dental root cells must respectively be collected by incision of the skin, perforation of the bone marrow, craniotomy, liposuction, surgical operation, tooth extraction and gingivectomy, all being strongly invasive methods. Therefore, these cells are not desirable as a raw material for human iPS cells. Hematopoietic progenitor cells can be collected not only by perforation of the bone marrow but also from peripheral blood after pretreatment such as administration of granulocyte colony-stimulating factor (G-CSF). However, such pretreatment involves compulsive proliferation of hematopoietic stem cells and thus may increase the risk of leukemia. Such pretreatment is undesirable from the viewpoint of safety. Further, the skin, hair root and dental root are directly exposed to the air and cells derived from these tissues are most likely to have been contaminated with environmental microorganisms, so they are not desirable as a raw material for human iPS cells.
Further, it is desirable that a living human tissue cell as a raw material for human iPS cells has the lowest possible levels of mutations resulting from damage added to genome information. This is a condition that need to be satisfied to avoid the risk of carcinogenesis, especially when human iPS cells are to be used for medical purposes. Among the above-listed human tissue cells, skin-derived fibroblast cells and hair root-derived keratinocytes are known to repeatedly undergo damage to the genome by UV rays and repair thereof (Ikehata, Environ. Mol. Mutagen., 41, 280-292, 2003). Thus, it is believed that these cells have a higher risk of mutation than other tissue cells and are not desirable as a raw material for human iPS cells.
Considering the above-described conditions, a living human tissue cell as a raw material for human iPS cells is desirably a cell contained in pheripheral blood which does not require a strongly invasive collection method or a risky pre-treatment and which can be easily obtained by blood collection of only about 10 mL as usually carried out at clinical sites. Examples of cells contained in the peripheral blood of healthy adults include, but are not limited to, lymphocytes (T cells, B cells and NK cells), granulocytes (neutrophils, basophils and eosinophils), monocytes, erythrocytes and platelets.
A living human tissue cell as a raw material for human iPS cells must be a cell that has genetic information necessary for maintaining iPS cells (nuclear genes and mitocondrial genes) and organelles (mitocondria, endoplasmic reticula, etc.). In view of this, erythrocytes and platelets which lack nucleus, and granulocytes (neutrophils, basophils and eosinophils) which almost lack mitocondria and endoplasmic reticula/Golgi apparatuses are not appropriate as a raw material for human iPS cells.
Further, in order to secure complete pluripotency, it is desirable that a living human tissue cell as a raw material for human iPS cells is a cell which has complete genetic information identical to that of a fertilized egg and which does not have irreversible recombinations, mutations and gene deletions in its genetic information in association with cell differentiation. Specific examples of cells which have irreversible recombinations, mutations and gene deletions in their genetic information include peripheral blood-derived T cells in which rereversible recombinations have occurred in T cell receptor genes and peripheral blood-derived B cells in which irreversible recombinations have occurred in antibody genes. This means that, in principle, only one type of T cell receptor or antibody can be produced from iPS cells prepared from peripheral blood-derived T cells or B cells; such iPS cells are unable to differentiate to hematopoietic stem cells which are required to have the capacity to produce a wide variety of T cells and B cells. Therefore, among the above-listed cells, peripheral blood-derived T cells and peripheral blood-derived B cells are undesirable as a raw material for human iPS cells.
Further, since human iPS cells are prepared on mouse or nonself human-derived feeder cells, it is desirable that a living human tissue cell as a raw material for human iPS cells does not have cytotoxicity to heterologous cells and allogeneic nonself cells. Therefore, among the above-listed cells, NK cells which recognize non-autologous cells and show non-specific cytotoxicity are undesirable as a raw material for human iPS cells.
On the other hand, among the above-listed cells, monocytes have an intact nucleus and mitocondria/organelles and retain complete genomic information that has not undergone irreversible changes such as recombination and deletion. Besides, methods for purification of monocytes have been established; they are Ficoll centrifugation in which blood cells are fractionated by difference in specific gravity and a purification method using anti-CD14 antibody bound magnetic beads. By combining these techniques, it is possible to recover a cell population of very high purity (98% or more) in a short period of time and in an aseptic manner. Thus, monocytes are the only human tissue cell that satisfies all of the above-described conditions as a raw material for superior human iPS cells. Therefore, establishing a method of preparing human iPS cells using monocytes as a raw material has critical significance in the attempt of putting pluripotent stem cells into practical use.
On the other hand, any of the cells currently used as a raw material for preparing human iPS cells proliferates through cell division under laboratory culture conditions, and it is believed that the ability to proliferate through cell division is necessary for preparation of human iPS cells (Hanna, et al., Nature, 462, 595-601, 2009). In contrast, monocytes are cells at the terminal stage of differentiation from hematopoietic stem cells and do not have proliferative capacity. Conditions to cause cell division in monocytes in vitro are not known. Therefore, at present, it is believed that preparation of iPS cells from monocytes is not easy to accomplish.
Peripheral blood-derived mononuclear cells obtained by partially purifying peripheral blood by Ficoll centrifugation are composed of lymphocytes (about 80%) and monocytes (about 20%). Therefore, the human iPS cells prepared from peripheral blood mononuclear cells according to the methods described in Loh, et al, Cell Stem Cell, 7, 15-19, 2010 and Staerk, et al., Cell Stem Cell, 7, 20-24, 2010 may probably contain monocytes-derived cells. In fact, the human iPS cells reported in Loh, et al, Cell Stem Cell, 7, 15-19, 2010 and Staerk, et al., Cell Stem Cell, 7, 20-24, 2010 contained iPS cells free from DNA recombination in T cell receptor or antibody gene, suggesting that these cells are derived from mononuclear cells other than T cells and B cells. However, it is unknown from what cell are derived the iPS cells that are believed to be non-T cells. No report has been published to date that shows the preparation of human iPS cells using purified monocytes.
It is known that the nature and safety of self-derived human iPS cells for use in cell replacement therapy are greatly affected by the method of preparation of such iPS cells. In the methods reported in Takahashi, et al., Cell, 131, 861-872, 2007 and Yu, et al., Science, 318, 1917-1920, 2007 that perform gene expression using a retroviral vector or lentiviral vector, the genes used in the preparation of human iPS cells are inserted into the chromosomes and remain therein This means that human iPS cells prepared by these techniques do not satisfy the condition of “pluripotent stem cells having genetic information identical to that of a patient” as a requirement for cell replacement therapy. In the human iPS cells prepared from peripheral blood mononuclear cells by a method using a lentiviral vector as described in Loh, et al., Cell Stem Cell, 7, 15-19, 2010 and Staerk, et al., Cell Stem Cell, 7, 20-24, 2010, genes used in the preparation of human iPS cells are also inserted into the chromosomes and remain therein. Therefore, the thus prepared human iPS cells do not satisfy the condition of “pluripotent stem cells having genetic information identical to that of a patient” as a requirement for in cell replacement therapy.
Further, this phenomenon of gene insertion evokes the following problems in terms of ensuring safety in cell replacement therapy. Briefly, when a foreign gene is inserted at non-specific positions on chromosomes, genes in the vicinity of the insertion site may be abnormally activated to cause adverse effects such as oncogenic transformation of cells. For example, it is known that when genes are inserted with a retroviral vector at non-specific positions on chromosomes of a human bone marrow stem cell that maintains replication competence over a long period of time, oncogenes whose transcription is inhibited in normal cells are abnormally activated due to the effect of inserted foreign genes, causing oncogenic transformation of cells to occur at high frequency (Hacein-Bey-Abina, et al., Science, 302, 415-419, 2003).
This phenomenon of gene insertion further evokes the following problems in terms of ensuring safety in cell replacement therapy. Briefly, in iPS cells prepared by inserting foreign genes into chromosomes, expression of the foreign genes is inhibited as long as the undifferentiated state is maintained. However, as the iPS cell differentiates to a tissue cell, expression of the foreign genes is induced and the tissue cell may turn cancerous. For example, the mouse individual derived from mouse iPS cells prepared by introducing four genes, Oct3/4, Sox2, Klf4 and c-Myc, into skin-derived normal fibroblast cells with a retroviral vector is known to develop cancer at high frequency as a result of reactivation of the foreign c-Myc gene introduced thereinto (see Takahashi and Yamanaka, Cell, 126, 663-676, 2006). In addition to expression of c-Myc gene, it has been pointed out that expression of Klf4 and Oct3/4 genes may also lead to oncogenic transformation of cells (Jaenishi and Young, Cell, 132, 567-582, 2008).
In order to solve the various problems resulting from gene insertion into chromosomes, human iPS cells must be prepared by a method that will not leave foreign genes on chromosomes. As one example of such technology, it has been reported that after preparing human iPS cells with four genes, Oct3/4, Sox2, Klf4 and c-Myc, being inserted at random into chromosomes, the inserted genes can be removed by introducing Cre recombinase (Woltjen, et al., Nature, 458, 766-770, 2009). This technology is also used in the preparation of iPS cells from peripheral blood mononuclear cells as disclosed in Staerk, et al., Cell Stem Cell, 7, 20-24, 2010. (However, removal of the reprogramming genes is not reported in Staerk, et al., Cell Stem Cell, 7, 20-24, 2010). According to the above technology, promoter regions necessary for expressing the reprogramming genes still remain on chromosomes. Therefore, the genetic information of the prepared iPS cells is not completely identical to the genetic information of a corresponding normal cell, and insertion mutation might possibly occur.
Yu et al. reported that seven genes, Oct3/4, Sox2, Klf4, c-Myc, Nanog, LIN28 and SV40 T antigen, were expressed simultaneously in human normal fibroblast cells using a circular DNA vector (EBV vector) having the replication origin of Epstein-Barr virus (EBV) and EBNA1 gene and being capable of extrachromosomal replication, and that foreign gene-free human iPS cells could be prepared utilizing spontaneous dropping off of the vector (see Yu, et al., Science, 324, 797-801, 2009). Recently, Fusaki et al. reported a method in which Oct3/4, Sox2, Klf4 and c-Myc genes were expressed in human skin-derived fibroblast cells or peripheral blood-derived T cells using Sendai virus as a vector capable of expressing foreign genes without inserting them into chromosomes, to thereby prepare pluripotent stem cells (Fusaki, et al., Proc. Jpn. Acad. Ser. B85, 348-362, 2009; Seki, et al., Cell Stem Cell, 7, 11-14, 2010; WO 2010/008054). In this method, four reprogramming genes were loaded on separate vectors, and mixed infection was carried out. It was reported that pluripotent stem cells were prepared at a maximum efficiency of 1%. Another method was reported in which synthetic mRNAs encoding Oct3/4, Sox2, Klf4 and c-Myc proteins were introduced into human fibroblast cells to thereby prepare foreign gene-free human iPS cells at a maximum efficiency of 2% at the highest (Warren, et al., Cell Stem Cell, 7, 1-13, 2010). However, neither of these methods is known to be capable of preparing human iPS cells from monocytes.
Accordingly, there has been reported no method that can be used to prepare human pluripotent stem cells required for cell replacement therapies or the like and which satisfies the following two conditions: 1) using, as a raw material, human peripheral blood-derived monocytes that retain complete genetic information and are yet obtainable by blood collection which is less invasive and has a smaller risk of contamination with microorganisms; and 2) being a technology that enables preparation of human pluripotent stem cells where no externally introduced reprogramming genes are left in order to secure safety.